The RNAprotect Bacteria Reagent is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

GeneChip analysis.

Total RNA was isolated from RNAprotect stabilized E. coli cultures using the RNeasy Protect Bacteria Kit (RNeasy Protect Bacteria) or from unstabilized cultures using a commercial precipitation method (Supplier EIII). To distinguish between gene expression under defined culture conditions and effects of artifactual gene induction during harvesting and RNA isolation, the RNA polymerase inhibitor rifampicin was added to half the culture prior to RNA isolation. Differences in transcript levels with and without rifampicin therefore generally reflect the degree of RNA degradation. [A] GeneChip analysis of E. coli microarrays was carried out according to standard Affymetrix protocols. [B] The percentage of genes expressed was estimated as the number of different transcripts determined present by GeneChip analysis divided by the total number of transcripts represented on the microarray. (Data from a collaborative study with Affymetrix.)

RNAprotect Bacteria Reagent procedure.

RNAprotect Bacteria Reagent prevents mRNA degradation.

To monitor RNA degradation only, transcription was stopped by adding the RNA polymerase inhibitor rifampicin to a growing culture of E. coli. The culture was divided into two halves, and RNAprotect Bacteria Reagent was added to one half. Samples were left at room temperature for 0, 4, 8, and 16 minutes before centrifugation and RNA isolation. The resulting RNA was analyzed by agarose gel electrophoresis (top panel). Expression of two marker genes with different half lives was examined by northern blot analysis. Middle panel: ompA (half life of 15 minutes); bottom panel: α-lactamase (half life of 2–5 minutes).

Performance

Use of RNAprotect Bacteria Reagent overcomes the problems of RNA degradation and changes in expression profiles by providing immediate stabilization prior to RNA isolation (see figures "RNAprotect Bacteria Reagent prevents mRNA degradation" and "GeneChip analysis"). RNAprotect Bacteria Reagent is suitable for use with a wide range of bacterial species, both Gram positive (e.g., Staphylococcus aureusand Mycobacterium avium) and Gram negative (e.g., Escherichia coliand Salmonella typhimurium). Bacteria can be grown in either minimal or complex medium prior to isolation of RNA.

Principle

Traditional methods for cell harvesting and RNA isolation can lead to vast changes in bacterial expression profiles. Enzymatic degradation of RNA leads to reduction or loss of many transcripts because bacterial mRNA molecules have very short half lives of only a few minutes. In addition, genes can be induced during handling and processing of samples, leading to higher expression levels of certain transcripts. Use of RNAprotect Bacteria Reagent overcomes these problems by providing immediate stabilization of RNA prior to RNA isolation procedures.

Procedure

Two volumes of reagent are added directly to 1 volume of bacterial culture prior to RNA isolation, providing immediate stabilization of RNA (see figure "RNAprotect Bacteria Reagent procedure"). The stabilization allows time for efficient bacterial lysis using a choice of protocols: enzymatic lysis, mechanical disruption, or a combination of both methods. QIAGEN recommends the TissueLyser for efficient mechanical disruption.

Applications

Bacteria stabilized in RNAprotect Bacteria Reagent can be used for total RNA isolation using RNeasy Kits.